Gut microbiome diversity may alter oral bioavailability and contribute to individual variations in drug therapy. Therefore, understanding molecular links between the gut microbiome and oral drug disposition is critical for realizing the goals of precision medicine. The purpose of the study was to characterize the function of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) using intestinal organoids derived from germ free and humanized mice. Typically, transport across the epithelial cell layers occurs by active transport and passive diffusion. Using mouse models that differ in bacterial content and fluorescence scanning, and live imaging techniques we studied the impact of bacteria on protein expression and function. The accumulation of membrane permeable acetoxymethyl calcein (calcein-AM), a widely used probe to monitor P-gp mediated efflux activity, in organoids was determined with and without the presence of P-gp inhibitors Cyclosporine A and LY335979. The uptake of calcein-AM was higher within the enterocytes of humanized mice colonoids compared to those obtained from germ free mice. These results correlated with immunohistochemistry on tissue explants that showed a lower expression of P-gp in germ free colon compared to the humanized mice colon. Both P-gp inhibitors showed an increase in calcein-AM uptake owing to the reduced transepithelial transport. Germ free colonoids showed a higher uptake of the BCRP probe Pheophorbide-A (Phe A) within the enterocytes as compared to humanized mice colonoids. These results correlated with Immunohistochemistry experiments that showed a lower expression of BCRP in germ free colon compared to humanized mice colon. These results demonstrate that intestinal organoids can be used as a powerful model to study expression and function of efflux transporters in the GI tract.
University of Minnesota M.S. thesis. May 2020. Major: Pharmaceutics. Advisor: Karunya Kandimalla. 1 computer file (PDF); iv, 38 pages
To Investigate The Impact Of Gut Bacteria On Efflux Transporter Expression And Function In Gastrointestinal Mucosae.
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